1. The Field of the Invention
The present invention lies in the technical field of cell biology and transplantation medicine. It concerns devices and methods for rapid and noninvasive analysis or checking of biological samples, especially for sterility control, for characterization of infectious particles and microorganisms contained in the biological sample and for characterization of tissue cells and transplants. The main areas of application of the invention are biotechnological production of pharmacologically active ingredients and therapeutic agents as well as transplantation medicine.
2. The Relevant Technology
The first aspect of the invention concerns contamination and quality control especially in transplantation medicine. In individual therapy of organ defects with organ-like tissue cultures, for example, skin or cartilage transplants, freedom from germs of the biological implants is a legally prescribed condition for release for implantation. The detection of contaminating microorganisms like bacteria and fungi is only possible with a time delay in the known detection methods. Sterility and contamination checks of biological implants have thus far occurred via known contact and smear samples of the fluids being investigated in subsequent incubation. A shortcoming is that the results of analysis with characterization of the contaminating microorganism, which is sometimes only a slowly growing pathogen, is only available about 2 weeks after release of the biological implant for implantation in the patient. Only then can corresponding therapeutic steps against the detected contamination be initiated, for example, specific antibiotic therapy. Means and methods are therefore desirable through which contamination of biological implants could be sensitively and selectively detected within a short time, and the existing contaminated microorganism characterized and, if necessary, identified.
For quality control of biological implants, tissue preparations or transplants, it is desirable to be able to characterize the culture tissue cells of the implant, for example, human cartilage cells, before application but also during culturing of the implant. The degree of differentiation and/or vitality of the cells should then be determined as a priority. This is of particularly great significance in implants from (autologous) human cells, since these cells can dedifferentiate during culturing, in which case they lose their tissue-specific properties desired for therapy. The quality of the implant diminishes on this account. Dedifferentiated cells also often have a tendency toward increased proliferation rate, for which reason they are viewed as potential cancer cells. Means and methods for characterization and typing of tissue cells and the extracellular matrix of the tissue and therefore for quality control of the biological implants are therefore desirable.
Quality control of biological implants is not uniformly regulated by law at present, since there is no known method that could be equally applied as gold standard to the wide variety of tissue cell types. It is known that immune cytological methods are used whose quality depends mostly on the specificity of the employed antibodies and which cannot be normalized or standardized. Known methods for characterization of biological material are “invasive” methods that make reuse of the investigated cells impossible. For example, in the known flow cytometry the cells are characterized by expression of certain surface molecules (CD antigen test). Based on this procedural deficiency only parts of the cultivated tissue can now be proposed as a biological implant for implantation. The other part must be “sacrificed” for invasive characterization of the cells. Means and methods are therefore desirable in which a noninvasive, nondestructive biological characterization of the cells being investigated can occur, if possible, in the liquid culture medium. It is also desirable to minimize the load on the tissue during the measurement.
Another aspect of the invention concerns sterility control in the production of drugs or active ingredients according to essentially biotechnological methods. In such methods active ingredients are produced by biological cells or cell systems, like microorganisms and/or tissue cells in so-called bioreactors. On an industrial scale bioreactors with volumes of 50 liters or more are used. Recovery of the active ingredients often requires culturing time in the reactors of several weeks. A multistage purification is generally connected to the bioreactor in the production chain, from which the desired active ingredient composition can ultimately be obtained.
It is then essential that no foreign contamination occur in the reactor charge and the sterility of the active ingredient composition recovered from the bioreactor is guaranteed. In known methods at the beginning and end of reactor culturing or at the end of the production change, the charge is checked for foreign contamination and sterility. In the case of contamination, the entire product volume must be discarded.
In known sterility tests biological samples are taken from the running production process in the form of small amounts of liquid, cultured according to conventional microbiological methods on a nutrient medium and then differentiated to determine the type of contamination. Results can only be expected with a delay of several days. A shortcoming is that the possibilities for regulatory intervention into the process are then limited. In most cases at least the employed resources for continuation of the production process between the time of liquid sampling and the time of positive detection of contamination are lost.
To improve the production process and avoid costs there is a requirement for continuous testing for sterility or contamination over the entire culturing time, during the entire production process and/or along the entire production chain. A rapid detection method is desirable that offers a short detection time and means to perform such a method. At the same time, high specificity and sensitivity should be present. Such a detection method should preferably also be suitable to clearly establish the type of contamination, i.e., especially to determine the contaminating microorganism. In addition to saving costs for the otherwise lost resources, a time saving would also be connected to this, since the contaminated process could be quickly terminated and a new process started in a timely fashion.
Raman spectroscopy is a known method that has been used thus far mostly in chemical analysis and surface characterization. The method is also being increasingly used for characterization of biological samples and in the investigation of biological fluids. When the biological sample is irradiated with excitation radiation of a certain wavelength or wavelength range, both elastic and inelastic scattering processes occur on the irradiated molecules in the sample. The main fraction of the introduced excitation light is elastically scattered in the sample (essentially so-called Rayleigh scattering). In addition, inelastic scattering to a smaller extent occurs on the molecules, so-called Raman scattered radiation. The Raman scattered radiation differs in energy content and therefore wavelength from the introduced excitation radiation. The Raman scattered radiation has both a fraction shifted toward shorter wavelengths (anti-Stokes lines) and a fraction of longer wavelengths (Stokes lines). The spectral distribution of the fraction of Raman scattered radiation shifted toward longer wavelengths is regularly understood as the Raman spectrum (Stokes lines). The Raman spectrum is mostly characteristic for the irradiated molecule or the molecule composition of the irradiated sample. With methods based mostly on statistical algorithms, such spectra can clearly be assigned to certain molecules of an irradiated sample so that molecule compositions, biological cells, cell compartments and some cellular structures can be characterized and if necessary specified.
In a method known from WO 2004/099763 A1 Raman spectroscopy is used to investigate and analyze biological cells in which the microorganisms being investigated are cultured, isolated, exposed to excitation radiation and the Raman scattered radiation emitted by the cells analyzed spectroscopically. From the recorded spectral distributions of the emitted Raman scattered radiation, the characteristic values for the corresponding microorganisms are recorded by statistical analyses, for example, certain bands in the spectrum, and compared with corresponding characteristics of known microorganisms stored in databases. The analysis method is invasive, since the microorganisms being investigated are released from the cell and prepared. Preculturing of the analyzed cells is not prescribed.
A drawback in this method is the invasive approach, which leads to destruction of the analyzed cells. Another drawback is the long measurement times connected with known Raman spectroscopic methods.
Starting from the prior art, the technical problem underlying the present invention consists mostly of providing means and methods for simple-to-handle, rapid, noninvasive, nondestructive detection and characterization of biological cells in biological samples, preferably cell combinations, tissues, cell suspensions or biological fluids.